Regulation of the Age-Dependent Activity of the FOXO Transcription Factor DAF-16 in Adult Caenorhabditis elegans Roundworms

The insulin and insulin-like growth factor signaling (IIS) pathway regulates stress resistance and contributes to longevity in evolutionarily diverse species. In the roundworm Caenorhabditis elegans the lifespan of daf-2 mutants, which lack functional insulin receptors, is twice that of wildtype animals. This phenotype is entirely dependent upon the FOXO transcription factor DAF-16. In the absence of negative regulation through the IIS pathway, DAF-16 upregulates genes important for stress resistance, defense, and repair. While it can be activated in response to environmental insults, in young animals DAF-16 is normally inhibited. However, we and others have demonstrated that the activity of DAF-16 increases in an age-dependent manner. The mechanism by which DAF-16 is regulated during aging has not yet been defined. Here we used a reverse genetic approach in C. elegans to investigate the function of genes that act upstream in the insulin signaling pathway to ask about their potential roles in modulating DAF-16 activity in post-reproductive adult animals. We tested the hypothesis that activation of DAF-2 and the ensuing activation of the PDK-1 kinase must be blocked in order to permit DAF-16 to regulate its transcriptional targets. By analyzing the expression of a GFP-labeled transcriptional reporter and by measuring gene expression levels by qRT-PCR, we found that in an age-dependent manner DAF-18, an ortholog of mammalian PTEN that prevents PDK-1 from being activated, is necessary for the increased expression of DAF-16 targets over time. Further, we found that RNAi targeting daf-18 reduced the ability of adult but not larval stage animals to resist infection by the bacterial pathogen Pseudomonas aeruginosa. This indicates that DAF-18 is required for DAF-16-mediated innate immunity later in life. Our observations raise the intriguing possibility that an agonist engages DAF-2 to initiate signaling through the IIS pathway at the same time that DAF-16 is activated during adulthood. Previous studies established a role for micro RNAs (miRNAs) in regulating DAF-2 activity, and we asked whether this could be true in the context of aging by knocking down the expression of alg-2, an argonaute protein involved in miRNA biogenesis. We reasoned that expression of a DAF-2 ligand might be controlled post-transcriptionally by miRNA. Our functional analysis implies a possible genetic interaction between alg-2 and daf-18 that is most evident in adult C. elegans, suggesting that their gene products may act in the same pathway that ultimately governs DAF-16 function. Together, our results reveal elements of a transcriptional regulatory mechanism that is specific to adult animals and that involves evolutionarily conserved components, suggesting its general relevance to the aging process in diverse organisms. © FASEB.